1. Field of the Invention
This invention relates to methods of facilitating the entry of biologically-active compounds into phagocytic cells and for targeting such compounds to specific organelles within the cell. The invention specifically provides compositions of matter and pharmaceutical embodiments of such compositions comprising conjugates of such biologically-active compounds covalently linked to particulate carriers generally termed microparticles. Particular embodiments of such compositions include compositions wherein the biologically-active compounds are antimicrobial drugs. In such compositions the antimicrobial drug is covalently linked to a microparticle via an organic linker molecule which is the target of a microorganism-specific protein having enzymatic activity. Thus, the invention provides cell targeting of drugs wherein the targeted drug is only released in cells infected with a particular microorganism. Alternative embodiments of such specific drug delivery compositions also contain polar lipid carrier molecules. Particular embodiments of such conjugates comprise antimicrobial drugs covalently linked both to a microparticle via an organic linker molecule and to a polar lipid compound, to facilitate targeting of such drugs to particular subcellular organelles within the cell.
2. Background of the Related Art
A major goal in the pharmacological arts has been the development of methods and compositions to facilitate the specific delivery of therapeutic and other agents to the appropriate cells and tissues that would benefit from such treatment, and the avoidance of the general physiological effects of the inappropriate delivery of such agents to other cells or tissues of the body. The most common example of the need for such specificity is in the field of antineoplastic therapy, in which the amount of a variety of anti-neoplastic agents that can be safely administered to a patient is limited by their cytotoxic effects.
It is also recognized in the medical arts that certain subcellular organelles are the sites of pharmacological action of certain drugs or are involved in the biological response to certain stimuli. Specific delivery of diagnostic or therapeutic compounds to such intracellular organelles is thus desirable to increase the specificity and effectiveness of such clinical diagnostic or therapeutic techniques.
A. Drug Targeting
It is desirable to increase the efficiency and specificity of administration of a therapeutic agent to the cells of the relevant tissues in a variety of pathological states. This is particularly important as relates to antimicrobial drugs. These drugs typically have pleiotropic antibiotic and cytotoxic effects that damage or destroy uninfected cells as well as infected cells. Thus, an efficient delivery system which would enable the delivery of such drugs specifically to infected cells would increase the efficacy of treatment and reduce the associated "side effects" of such drug treatments, and also serve to reduce morbidity and mortality associated with clinical administration of such drugs.
Numerous methods for enhancing the cytotoxic activity and the specificity of antimicrobial drug action have been proposed. One method, receptor targeting, involves linking the therapeutic agent to a ligand which has an affinity for a receptor expressed on the desired target cell surface. Using this approach, an antimicrobial agent or drug is intended to adhere to the target cell following formation of a ligand-receptor complex on the cell surface. Entry into the cell could then follow as the result of internalization of ligand-receptor complexes. Following internalization, the antimicrobial drug may then exert its therapeutic effects directly on the cell.
One limitation of the receptor targeting approach lies in the fact that there are only a finite number of receptors on the surface of target cells. It has been estimated that the maximum number of receptors on a cell is approximately one million (Darnell et al., 1986, Molecular Cell Biology, 2d ed., W. H. Freeman: New York, 1990). This estimate predicts that there may be a maximum one million drug-conjugated ligand-receptor complexes on any given cell. Since not all of the ligand-receptor complexes may be internalized, and any given ligand-receptor system may express many-fold fewer receptors on a given cell surface, the efficacy of intracellular drug delivery using this approach is uncertain. Other known intracellular ligand-receptor complexes (such as the steroid hormone receptor) express as few as ten thousand hormone molecules per cell. Id. Thus, the ligand-receptor approach is plagued by a number of biological limitations.
Other methods of delivering therapeutic agents at concentrations higher than those achievable through the receptor targeting process include the use of lipid conjugates that have selective affinities for specific biological membranes. These methods have met with little success. (see, for example, Remy et al., 1962, J. Org. Chem. 27: 2491-2500; Mukhergee & Heidelberger, 1962, Cancer Res. 22: 815-22; Brewster et al., 1985, J. Pharm. Sci. 77: 981-985).
Liposomes have also been used to attempt cell targeting. Rahman et al., 1982, Life Sci. 31: 2061-71 found that liposomes which contained galactolipid as part of the lipid appeared to have a higher affinity for parenchymal cells than liposomes which lacked galactolipid. To date, however, efficient or specific drug delivery has not been predictably achieved using drug-encapsulated liposomes. There remains a need for the development of cell-specific and organelle-specific targeting drug delivery systems.
B. Phagocytic Cell-Specific Targeting
Cell-specific targeting is also an important goal of antimicrobial therapy, particularly in the event that a specific cell type is a target of acute or chronic infection. Targeting in the case of infection of a specific cell type would be advantageous because it would allow administration of biologically-toxic compounds to an animal suffering from infection with a microbial pathogen, without the risk of non-specific toxicity to uninfected cells that would exist with nontargeted administration of the toxic compound. An additional advantage of such targeted antimicrobial therapy would be improved pharmacokinetics that would result from specific concentration of the antimicrobial agent to the sites of infection, i.e., the infected cells.
Phagocytic cells such as monocytes and macrophages are known to be specific targets for infection of certain pathogenic microorganisms.
Sturgill-Koszycki et al., 1994, Science 263: 678-681 disclose that the basis for lack of acidification of phagosomes in M. avium and M. tuberculosis-infected macrophages is exclusion of the vesicular proton-ATPase.
Sierra-Honigman et al., 1993, J. Neuroimmunol. 45: 31-36 disclose Borna disease virus infection of monocytic cells in bone marrow.
Maciejewski et al., 1993, Virol. 195: 327-336 disclose human cytomegalovirus infection of mononucleated phagocytes in vitro.
Alvarez-Dominguez et al., 1993, Infect. Immun. 61: 3664-3672 disclose the involvement of complement factor Clq in phagocytosis of Listeria monocytogenes by macrophages.
Kanno et al., 1993, J. Virol. 67: 2075-2082 disclose that Aleutian mink disease parvovirus replication depends on differentiation state of the infected macrophage.
Kanno et al., 1992, J. Virol. 66: 5305-5312 disclose that Aleutian mink disease parvovirus infects peritoneal macrophages in mink.
Narayan et al., 1992, J. Rheumatol. 32: 25-32 disclose arthritis in animals caused by infection of macrophage precursors with lentivirus, and activation of quiescent lentivirus infection upon differentiation of such precursor cells into terminally-differentiated macrophages.
Horwitz, 1992, Curr. Top. Microbiol. Immunol. 181: 265-282 disclose Legionella pneumophila infections of aveolar macrophages as the basis for Legionnaire's disease and Pontiac fever.
Sellon et al., 1992, J. Virol. 66: 5906-5913 disclose equine infectious anemia virus replicates in tissue macrophages in vivo.
Groisman et al., 1992, Proc. Natl. Acad. Sci. USA 89: 11939-11943 disclose that S. typhimurium survives inside infected macrophages by resistance to antibacterial peptides.
Friedman et al., 1992, Infect. Immun. 60: 4578-4585 disclose Bordetella pertussis infection of human macrophages.
Stellrecht-Broomhall, 1991, Viral Immunol. 4: 269-280 disclose that lymphocytic choriomeningitis virus infection of macrophages promotes severe anemia caused by macrophage phagocytosis of red blood cells.
Frehel et al., 1991, Infect. Immun. 59: 2207-2214 disclose infection of spleen and liver-specific inflammatory macrophages by Mycobacterium avium, the existence of the microbe in encapsulated phagosomes within the inflammatory macrophages and survival therein in phagolysosomes.
Bromberg et al., 1991, Infect. Immun. 59: 4715-4719 disclose intracellular infection of alveolar macrophages.
Mauel, 1990, J. Leukocyte Biol. 47: 187-193 disclose that Leishmania spp. are intracellular parasites in macrophages.
Buchmeier and Heffron, 1990, Science 248: 730-732 disclose that Salmonella typhimurium infection of macrophages induced bacterial stress proteins.
Panuska et al., 1990, J. Clin. Invest. 86: 113-119 disclose productive infection of alveolar macrophages by respiratory syncytial virus.
Cordier et al., 1990, Clin. Immunol. Immunopathol. 55: 355-367 disclose infection of alveolar macrophages by visna-maedi virus in chronic interstitial lung disease in sheep.
Schlessinger and Horwitz, 1990, J. Clin. Invest. 85: 1304-1314 disclose Mycobacterium leprae infection of macrophages.
Clarke et al., 1990, AIDS 4: 1133-1136 disclose human immunodeficiency virus infection of alveolar macrophages in lung.
Baroni et al., 1988, Am. J. Pathol. 133: 498-506 disclose human immunodeficiency virus infection of lymph nodes.
Payne et al, 1987, J. Exp. Med. 166: 1377-1389 disclose Mycobactertium tuberculosis infection of macrophages.
Murray et al., 1987, J. Immunol. 138: 2290-2296 disclose that liver Kupffer cells are the initial targets for L. donovani infection.
Koenig et al., 1986, Science 233: 1089-1093 disclose human immunodeficiency virus infection of macrophages in the central nervous system.
Horwitz and Maxfield, 1984, J. Cell Biol. 99: 1936-1943 disclose that L. pneumophila survives in infected phagocytic cells at least in pan by inhibiting reduction of intraphagosomic hydrogen ion concentration (pH).
Shanley and Pesanti, 1983, Infect. Immunol. 41: 1352-1359 disclose cytomegalovirus infection of macrophages in murine cells.
Horwitz, 1983, J. Exp. Med. 158: 2108-2126 disclose that L. pneumophila is an obligate intracellular parasite that is phagocytized into a phagosome wherein fusion with lysosome is inhibited.
Chang, 1979, Exp. Parisitol. 48: 175-189 disclose Leischmania donovani infection of macrophages.
Wyrick and Brownridge, 1978, Infect. Immunol. 19: 1054-1060 disclose Chlamydia psittaci infection of macrophages.
Nogueira and Cohn, 1976, J. Exp. Med. 143: 1402-1420 disclose Trypanosoma cruzi infection of macrophages.
Jones and Hirsch, 1972, J. Exp. Med. 136: 1173-1194 disclose Toxoplasma gondii infection of macrophages.
Persistent infection of phagocytic cells has been reported in the prior art.
Embretson et al., 1993, Nature 362: 359-361 disclose covert infection of macrophages with HIV and dissemination of infected cells throughout the immune system early in the course of disease.
Schnorr et al., 1993, J. Virol. 67: 4760-4768 disclose measles virus persistent infection in vitro in a human monocytic cell line.
Meltzer and Gendelman, 1992, Curr. Topics Microbiol. Immunol. 181: 239-263 provide a review of HIV infection of tissue macrophages in brain, liver, lung, skin, lymph nodes, and bone marrow, and involvement of macrophage infection in AIDS pathology.
Blight et al., 1992, Liver 12: 286-289 disclose persistent infection of liver macrophages (Kuppfer cells) by hepatitis C virus.
McEntee et al., 1991, J. gen. Virol. 72: 317-324 disclose persistent infection of macrophages by HIV resulting in destruction of T lymphocytes by fusion with infected macrophages, and that the macrophages survive fusion to kill other T lymphocytes.
Kondo et al., 1991, J. gen. Virol. 72: 1401-1408 disclose herpes simplex virus 6 latent infection of monocytes activated by differentiation into macrophages.
King et al., 1990, J. Virol. 64: 5611-5616 disclose persistent infection of macrophages with lymphocytic choriomeningitis virus.
Schmitt et al., 1990, Res. Virol. 141: 143-152 disclose a role for HIV infection of Kupffer cells as reservoirs for HIV infection.
Gendelman et al., 1985, Proc. Natl. Acad. Sci. USA 82: 7086-7090 disclose lentiviral (visna-maedi) infection of bone marrow precursors of peripheral blood monocytes/macrophages that provide a reservoir of latently-infected cells.
Halstead et al., 1977, J. Exp. Med. 146: 201-217 disclose that macrophages are targets of persistent infection with dengue virus.
Mauel et al., 1973, Nature New Biol. 244: 93-94 disclose that lysis of infected macrophages with sodium dodecyl sulfate could release live microbes.
Attempts at drug targeting have been reported in the prior art.
Rubinstein et al., 1993, Pharm. Res. 10: 258-263 report colon targeting using calcium pectinate (CaPec)-conjugated drugs, based on degradation of CaPec by colon specific (i.e., microflora-specific) enzymes and a hydrophobic drug incorporated into the insoluble CaPec matrices.
Sintov et al., 1993, Biomaterials 14: 483-490 report colon-specific targeting using conjugation of drug to insoluble synthetic polymer using disaccharide cleaved by enzymes made by intestinal microflora, specifically, .beta.-glycosidic linkages comprising dextran.
Franssen et al., 1992, J. Med. Chem. 35: 1246-1259 report renal cell/kidney drug targeting using low molecular weight proteins (LMWP) as carriers, using enzymatic/chemical hydrolysis of a spacer molecule linking the drug and LMWP carrier.
Bai et al., 1992, J. Pharm. Sci. 81: 113-116 report intestinal cell targeting using a peptide carrier-drug system wherein the conjugate is cleaved by an intestine-specific enzyme, prolidase.
Gaspar et al., 1992, Ann. Trop. Med. Parasitol. 86: 41-49 disclose primaquine-loaded polyisohexylcyanoacrylate nanoparticles used to target Leschmania donovani infected macrophage-like cells in vitro.
Pardridge, 1992, NIDA Res. Monograph 120: 153-168--opioid-conjugated chimeric peptide carriers for targeting to brain across the blood-brain barrier.
Bai and Amidon, 1992, Pharm. Res. 9: 969-978 report peptide-drug conjugates for oral delivery and intestinal mucosal targeting of drugs.
Ashborn et al., 1991, J. Infect. Dis. 163: 703-709 disclose the use of CD4-conjugated Pseudomonas aeruginosa exotoxin A to kill HIV-infected macrophages.
Larsen et al., 1991, Acta Pharm. Nord. 3: 41-44 report enzyme-mediated release of drug from dextrin-drug conjugates by microflora-specific enzymes for colon targeting.
Faulk et al., 1991, Biochem. Int. 25: 815-822 report adriamycin-transferrin conjugates for tumor cell growth inhibition in vitro.
Zhang and McCormick, 1991, Proc. Natl. Acad. Sci. USA 88: 10407-10410 report renal cell targeting using vitamin B6-drug conjugates.
Blum et al., 1982, Int. J. Pharm. 12: 135-146 report polystyrene microspheres for specific delivery of compounds to liver and lung.
Trouet et al., 1982, Proc. Natl. Acad. Sci. USA 79: 626-629 report that daunorubicin-conjugated to proteins were cleaved by lysosomal hydrolases in vivo and in vitro.
Shen et al., 1981, Biochem. Biophys. Res. Commun. 102: 1048-1052 report pH-labile N-cis-acontinyl spacer moieties.
Monoclonal antibodies have been used in the prior art for drug targeting.
Serino et al, U.S. Pat. No. 4,793,986, issued Dec. 27, 1988, provides platinum anticancer drugs conjugated to polysaccharide (dextrin) career for conjugation to monoclonal antibodies for tumor cell targeting.
Bickel et al., 1993, Proc. Natl. Acad. Sci. USA 90: 2618-2622 discloses the use of a chimeric protein vector for targeting across blood-brain barrier using anti-transferrin monoclonal antibody.
Rowlinson-Busza and Epenetos, 1992, Curr. Opin. Oncol. 4: 1142-1148 provides antitumor immunotargeting using toxin-antibody conjugates.
Blakey, 1992, Acta Oncol. 31: 91-97 provides a review of antitumor antibody targeting of antineoplastic drugs.
Senter et al., 1991, in Immunobiology of Peptides and Proteins, Vol. VI, pp.97-105 discloses monoclonal antibodies linked to alkaline phosphatase or penicillin-V amidase to activate prodrugs specifically at site of antibody targeting, for therapeutic treatment of solid tumors.
Drug-career conjugates have been used in the prior art to provide time-release drug delivery agents,
Couveur and Puisieux, 1993, Adv. Drug Deliv. Rev. 10: 141-162 provide a review of microcapsule (vesicular), microsphere (dispersed matrix) and microparticle (1-250 .mu.m)-based drug delivery systems, based on degradation of particle with drug release, to provide time release of drugs, oral delivery via transit through the intestinal mucosa and delivery to Kupffer cells of liver.
Duncan, 1992, Anticancer Drugs 3: 175-210 provide a review of improved pharmokinetic profile of in vivo drug release of anticancer drugs using drug-polymer conjugates.
Heinrich et al., 1991, J. Pharm. Pharmacol. 43: 762-765 disclose poly-lactideglycolide polymers for slow release of gonadotropin releasing hormone agonists as injectable implants.
Wada et al. 1991, J. Pharm. Pharmacol. 43: 605-608 disclose sustained-release drug conjugates with lactic acid oligomers.
Specifically, polymer-conjugated drugs have been reported in the prior art, and attempts to adapt particulate conjugates have also been reported.
Ryser et al., U.S. Pat. No. 4,847,240, issued Jul. 11, 1989, provides cationic polymers for conjugation to compounds that are poorly transported into cells. Examples include the antineoplastic drug methotrexate conjugated with polylysine and other polycationic amino acids are the carriers.
Ellestad et al., U.S. Pat. No. 5,053,394, issued Oct. 1, 1991, provides carrier-drug conjugates of methyltrithiol antibacterial and antitumor agents with a spacer linked to a targeting molecule which is an antibody or fragment thereof, growth factors or steroids.
Kopecek et al., U.S. Pat. No. 5,258,453, issued Nov. 2, 1993, provides antitumor compositions comprising both an anticancer drug and a photoactivatable drug attached to a copolymeric carrier by functional groups labile in cellular lysosomes, optionally containing a targeting moiety that are monoclonal antibodies, hormones, etc.
Negre et al., 1992, Antimicrob. Agents and Chemother. 36: 2228-2232 disclose the use of neutral mannose-substituted polylysine conjugates with an anti-leischmanial drug (allopurinol riboside) to treat murine infected macrophages in vitro.
Yatvin, 1991, Select. Cancer. Therapeut. 7: 23-28 discusses the use of particulate carriers for drug targeting.
Hunter et al., 1988, J. Pharm. Phamacol. 40: 161-165 disclose liposome-mediated delivery of anti-leischmanial drugs to infected murine macrophages in vitro.
Saffran et al., 1986, Science 233: 1081-1084 disclose drug release from a particulate carder in the gut resulting from degradation of the carrier by enzymes produced by intestinal microflora.